Structural annotation of acylcarnitines detected in SRM 1950 using collision-induced dissociation and...

Acylcarnitines are important intermediates in fatty acid metabolism, shuttling acyl groups into mitochondria for β-oxidation and energy production. As biomarkers, their concentrations are utilized to diagnose metabolic and cardiovascular diseases, insulin resistance, and neurodegenerative disorders. However, in-depth structural characterization of acylcarnitines is limited by conventional collision-induced dissociation (CID), which yields fragments predominantly from the carnitine headgroup with minimal information regarding the fatty acyl chains. In this case, we employed a novel quadrupole time-of-flight (QToF) mass spectrometer, the Sciex ZenoTOF 8600, with CID and electron-induced dissociation (EID) to carry out in-depth analysis of acylcarnitines detected in National Institute of Standards and Technology (NIST) SRM 1950 reference plasma. With increased sensitivity and reduced accumulation times (95 ms) on the 8600 platform, we were able to confidently annotate 35 acylcarnitines, including isomeric species and functional groups such as hydroxylations and double bonds. EID provided comprehensive structural information, enabling the discrimination of isomers such as valeryl-, isovaleryl-, and 2-methylbutyryl-carnitine (Car 5:0), as well as the positioning of methyl branching via diagnostic fragments. Hydroxylated species, such as Car 16:0;3OH, were confirmed by the presence of diagnostic ions and matched to reference standards by retention time. Moreover, EID enabled the localization of double bonds within unsaturated species (e.g., Car 18:1, Car 18:2) via fragmentation patterns that are indicative of unsaturation positions, following established lipid fragmentation mechanisms. This work demonstrates that EID offers significant advancements for the structural elucidation of acylcarnitines, delivering enhanced sensitivity and deeper insights into isomeric and functional diversity.